NOTE: Indra - we need to make a truncated version of your for the pNIC-Bsa4 (Version3 cloning) because there is a transmembrane region on the edge of the protein (see http://www.cbs.dtu.dk/services/TMHMM/) We can do this by changing the primers for Version 3 with the tails. We can still use the pUC19 clone that you have.
Organism: Shigella flexneri Target:
Apyrase Phosphotase NCBI Gene # or RefSeq#:
876677 EC#:
3.6.1.5 PDB # or closest PDB entry is using homology model:
1D2T
Background/Disease: Shigella flexneri is a species of Gram-negative bacteria in the genus Shigell that can cause diarrhea in humans. There are several different serogroups of Shigella; S. flexneri belongs to group B. S. flexneri infections can usually be treated with antibiotics although some strains have become resistant. Less severe cases are not usually treated because they become more resistant in the future.
S. flexneri contians a virulence plasmid that codes for three virulence factors: a type-3 secretion system (T3SS), invasion plasmid antigen proteins (ipa proteins), and IcsA (used for cell-to-cell spread). Upon infection, S. flexneri injects the host cell cytoplasm with ipa proteins using the T3SS--a needle-and-syringe like apparatus common to many gram negative pathogens. These ipa proteins induce "membrane ruffling" by the host cell. Membrane ruffling creates membrane pockets which capture and engulf the bacteria. Once inside, S. flexneri utilizes host cell actin for propulsion to move directly from cell to cell using a cellular mechanism known as paracytophagy, similarly to the bacterial pathogen Listeria monocytogenes
Image of protein (PyMol or etc): Codon Optimized Sequence:
ATGAAAACCAAAAACTTCCTGCTGTTTTGCATTGCGACCAATATGATCTTCATCCCGTCTGCGAACGCCCTGAAAGCAGAAGGT
TTCCTCACCCAGCAGACCTCTCCGGATTCTCTGTCTATCCTGCCGCCACCGCCAGCGGAGGATTCTGTTGTTTTCCTGGCGGAC
AAAGCGCACTACGAATTTGGTCGTTCTCTCCGTGACGCGAACCGCGTTCGTCTGGCGTCTGAAGATGCGTACTACGAGAACTTC
GGTCTCGCCTTCTCTGACGCGTACGGTATGGACATCTCTCGTGAAAACACCCCGATCCTGTACCAGCTGCTGACCCAGGTACTG
CAGGACTCTCATGACTATGCTGTTCGCAACGCTAAAGAATACTACAAACGTGTTCGCCCGTTCGTTATCTACAAAGACGCGACCT
GCACCCCGGATAAAGACGAAAAAATGGCGATCACCGGTTCTTACCCGTCCGGTCACGCGAGCTTCGGTTGGGCGGTTGCGCTG
ATCCTGGCAGAAATCAACCCGCAGCGTAAAGCGGAAATCCTGCGTCGTGGTTACGAGTTCGGTGAATCTCGTGTTATCTGCGGC
GCACACTGGCAGTCCGACGTTGAAGCGGGTCGTCTGATGGGTGCGTCTGTAGTTGCGGTGCTGCACAACACGCCGGAATTCAC
CAAATCTCTGAGCGAAGCGAAGAAAGAATTCGAAGAACTGAACACTCCGACCAACGAACTGACCCCGTAA
Primer design results for pNIC-Bsa4 cloning: Forward Primer:
5' TACTTCCAATCCATGAAAACCAAAAACTTCCT 3'
32 bp
GC Content: 34.4%
0mM Mg: 59.2 C
1.5mM Mg: 67.2 C
2mM Mg: 67.8 C
4mM Mg: 68.9 C
6mM Mg: 69.5 C
Reverse Primer:
5' TATCCACCTTTACTGTTACGGGGTCAGTTCGTTGG '3
35 bp
GC Content: 48.6%
0mM Mg: 64.8 C
1.5mM Mg: 71.8 C
2mM Mg: 72.2 C
4mM Mg: 73.1 C
6mM Mg: 73.5 C
Organism:
Shigella flexneri
Target:
Apyrase Phosphotase
NCBI Gene # or RefSeq#:
876677
EC#:
3.6.1.5
PDB # or closest PDB entry is using homology model:
1D2T
Background/Disease:
Shigella flexneri is a species of Gram-negative bacteria in the genus Shigell that can cause diarrhea in humans. There are several different serogroups of Shigella; S. flexneri belongs to group B. S. flexneri infections can usually be treated with antibiotics although some strains have become resistant. Less severe cases are not usually treated because they become more resistant in the future.
S. flexneri contians a virulence plasmid that codes for three virulence factors: a type-3 secretion system (T3SS), invasion plasmid antigen proteins (ipa proteins), and IcsA (used for cell-to-cell spread). Upon infection, S. flexneri injects the host cell cytoplasm with ipa proteins using the T3SS--a needle-and-syringe like apparatus common to many gram negative pathogens. These ipa proteins induce "membrane ruffling" by the host cell. Membrane ruffling creates membrane pockets which capture and engulf the bacteria. Once inside, S. flexneri utilizes host cell actin for propulsion to move directly from cell to cell using a cellular mechanism known as paracytophagy, similarly to the bacterial pathogen Listeria monocytogenes
Image of protein (PyMol or etc):
Codon Optimized Sequence:
ATGAAAACCAAAAACTTCCTGCTGTTTTGCATTGCGACCAATATGATCTTCATCCCGTCTGCGAACGCCCTGAAAGCAGAAGGT
TTCCTCACCCAGCAGACCTCTCCGGATTCTCTGTCTATCCTGCCGCCACCGCCAGCGGAGGATTCTGTTGTTTTCCTGGCGGAC
AAAGCGCACTACGAATTTGGTCGTTCTCTCCGTGACGCGAACCGCGTTCGTCTGGCGTCTGAAGATGCGTACTACGAGAACTTC
GGTCTCGCCTTCTCTGACGCGTACGGTATGGACATCTCTCGTGAAAACACCCCGATCCTGTACCAGCTGCTGACCCAGGTACTG
CAGGACTCTCATGACTATGCTGTTCGCAACGCTAAAGAATACTACAAACGTGTTCGCCCGTTCGTTATCTACAAAGACGCGACCT
GCACCCCGGATAAAGACGAAAAAATGGCGATCACCGGTTCTTACCCGTCCGGTCACGCGAGCTTCGGTTGGGCGGTTGCGCTG
ATCCTGGCAGAAATCAACCCGCAGCGTAAAGCGGAAATCCTGCGTCGTGGTTACGAGTTCGGTGAATCTCGTGTTATCTGCGGC
GCACACTGGCAGTCCGACGTTGAAGCGGGTCGTCTGATGGGTGCGTCTGTAGTTGCGGTGCTGCACAACACGCCGGAATTCAC
CAAATCTCTGAGCGAAGCGAAGAAAGAATTCGAAGAACTGAACACTCCGACCAACGAACTGACCCCGTAA
CDS Gene Sequence Non-Codon Optimized:
ATGAAAACCAAAAACTTTCTTCTTTTTTGTATTGCTACAAATATGATTTTTATCCCCTCAGCAAATGCTC TGAAGGCAGAAGGTTTTCTCACTCAACAAACTTCACCAGACAGTTTGTCAATACTTCCGCCGCCTCCGGC AGAGGATTCAGTAGTATTTCTGGCTGACAAAGCTCATTATGAATTCGGCCGCTCGCTCCGGGATGCTAAT CGTGTACGTCTCGCTAGCGAAGATGCATACTACGAGAATTTTGGTCTTGCATTTTCAGATGCTTATGGCA TGGATATTTCAAGGGAAAATACCCCAATCTTATATCAGTTGTTAACACAAGTACTACAGGATAGCCATGA TTACGCCGTGCGTAACGCCAAAGAATATTATAAAAGAGTTCGTCCATTCGTTATTTATAAAGACGCAACC TGTACACCTGATAAAGATGAGAAAATGGCTATCACTGGCTCTTATCCCTCTGGTCATGCATCCTTTGGTT GGGCAGTAGCACTGATACTTGCGGAGATTAATCCTCAACGTAAAGCGGAAATACTTCGACGTGGATATGA GTTTGGAGAAAGTCGGGTCATCTGCGGTGCGCATTGGCAAAGCGATGTAGAGGCTGGGCGTTTAATGGGA GCATCGGTTGTTGCAGTACTTCATAATACACCTGAATTTACCAAAAGCCTTAGCGAAGCCAAAAAAGAGTTTGAAGAATTAAATACTCCTACCAATGAACTGACCCCATAA
Updated CDS Gene Sequence after being Cut with only 681 bp Non-Codon Optimized:
TACTTCCAATCCATGGCAGAAGGTTTTCTCACTCAACAAACTTCACCAGACAGTTTGTCAATACTTCCGCCGCCTCCGGCAGAGGATTCAGTAGTATTTCTGGCTGACAAAGCTCATTATGAATTCGGCCGCTCGCTCCGGGATGCTAATCGTGTACGTCTCGCTA
GCGAAGATGCATACTACGAGAATTTTGGTCTTGCATTTTCAGATGCTTATGGCATGGATATTTCAAGGGAAAATACCCCAATCT
TATATCAGTTGTTAACACAAGTACTACAGGATAGCCATGATTACGCCGTGCGTAACGCCAAAGAATATTATAAAAGAGTTCGTC
CATTCGTTATTTATAAAGACGCAACCTGTACACCTGATAAAGATGAGAAAATGGCTATCACTGGCTCTTATCCCTCTGGTCATGC
ATCCTTTGGTTGGGCAGTAGCACTGATACTTGCGGAGATTAATCCTCAACGTAAAGCGGAAATACTTCGACGTGGATATGAGTT
TGGAGAAAGTCGGGTCATCTGCGGTGCGCATTGGCAAAGCGATGTAGAGGCTGGGCGTTTAATGGGAGCATCGGTTGTTGCA
GTACTTCATAATACACCTGAATTTACCAAAAGCCTTAGCGAAGCCAAAAAAGAGTTTGAAGAATTAAATACTCCTACCAATGAACTGACCCCATAA
Codon Optimized Amino Acid Sequence:
MKTKNFLLFCIATNMIFIPSANALKAEGFLTQQTSPDSLSILPPPPAEDSVVFLADKAHY
EFGRSLRDANRVRLASEDAYYENFGLAFSDAYGMDISRENTPILYQLLTQVLQDSHDYAV
RNAKEYYKRVRPFVIYKDATCTPDKDEKMAITGSYPSGHASFGWAVALILAEINPQRKAE
ILRRGYEFGESRVICGAHWQSDVEAGRLMGASVVAVLHNTPEFTKSLSEAKKEFEELNTP
TNELTP*
Amino Acid Sequence:
>gi|13448943|ref|NP_085159.1| apyrase [Shigella flexneri 5a] MKTKNFLLFCIATNMIFIPSANALKAEGFLTQQTSPDSLSILPPPPAEDSVVFLADKAHYEFGRSLRDAN RVRLASEDAYYENFGLAFSDAYGMDISRENTPILYQLLTQVLQDSHDYAVRNAKEYYKRVRPFVIYKDAT CTPDKDEKMAITGSYPSGHASFGWAVALILAEINPQRKAEILRRGYEFGESRVICGAHWQSDVEAGRLMG ASVVAVLHNTPEFTKSLSEAKKEFEELNTPTNELTP
~52% homology to a Escherichia blattae acid phosphatase 1D2T on PDB
Updated Amino Acid Sequence after being Cut:
MYFQSMAEGFLTQQTSPDSLSILPPPPAEDSVVFLADKAHYEFGRSLRDANRVRLASEDAY YENFGLAFSDAYGMDISRENTPILYQLLTQVLQDSHDYAVRNAKEYYKRVRPFVIYKDAT CTPDKDEKMAITGSYPSGHASFGWAVALILAEINPQRKAEILRRGYEFGESRVICGAHWQ SDVEAGRLMGASVVAVLHNTPEFTKSLSEAKKEFEELNTPTNELTP*Primer design results for pNIC-Bsa4 cloning:
Forward Primer:
5'
TACTTCCAATCCATGAAAACCAAAAACTTCCT 3'
32 bp
GC Content: 34.4%
0mM Mg: 59.2 C
1.5mM Mg: 67.2 C
2mM Mg: 67.8 C
4mM Mg: 68.9 C
6mM Mg: 69.5 C
Reverse Primer:
5'
TATCCACCTTTACTGTTACGGGGTCAGTTCGTTGG '3
35 bp
GC Content: 48.6%
0mM Mg: 64.8 C
1.5mM Mg: 71.8 C
2mM Mg: 72.2 C
4mM Mg: 73.1 C
6mM Mg: 73.5 C
Actual Primers ordered in July:
Updated Primers ordered in September:
Virtual Screening:
RMS = 4.806 (206 to 206 atoms)